Correlative Light and Electron Microscopy: Methods and Applications


Light has its limits, and even in the world of ‘super resolution’ microscopy, many cellular structures and even large protein complexes are not resolvable in the light microscope – although it is ideal for watching living cells and tissues and linking structural changes to functional outcomes. Electron microscopy can yield molecular level resolution but is limited to nonliving samples that have been extensively processed to preserve structure and be visible using electron optics. While it would be ideal to have a single system which provided nanoscale information on living samples, until that day arrives, the obvious answer is to use both light microscopy and electron microscopy as complementary approaches. Localising the same labelled structure in both imaging modalities is known as correlative LM and EM or correlated light and electron microscopy. Various methods and instrumentation are used for correlating data from the same sample imaged using both light and electrons.

Key Concepts

  • Light microscopes cannot resolve specimens at the nanoscale.
  • Combining light and electron microscopy can link ultrastructure to function.
  • For CLEM, exactly the same region of interest (ROI) is imaged in both the light and electron microscope.
  • 3D (volume) electron microscopy can provide spatial information at the nanoscale.
  • Using special probes, proteins can be located in both light and electron microscopes.

Keywords: correlative light and electron microscopy; light microscopy; electron microscopy; immunochemistry; horse radish peroxidase; fluorescence; photooxidation; APEX

Figure 1. In‐resin fluorescence. CLEM of GFP‐C1 and mCherry‐H2B in HeLa cells using postembedding light microscopy. GFP‐C1 fluorescent signals overlaid onto electron micrographs of the corresponding region using the IRF method. (a) Image of GFP‐C1 signal alone imaged under dry conditions, and overlaid directly onto the matching electron micrograph, for a cell embedded in HM20. The fluorescent signal corresponds to the nuclear envelope, and the other structures within the cytoplasm. (b) An adjacent section through the cell shown in (a). (c–e) Increased precision of GFP‐C1 localisation to Golgi stacks (g) and the NE. In (c), black arrows indicate localisation of fluorescent signal to the highly curved tips of Golgi cisternae. An enlarged view from an adjacent section is shown in (d). Localisation to the NE is also evident. In (e), white arrows indicate localisation of fluorescent signal to the nucleoplasmic reticulum. (f, g) Localisation of GFP‐C1 to endoplasmic reticulum and to membrane stacks (black arrows) in cells expressing low (f) and high (g) levels of the GFP‐C1 construct. ER, endoplasmic reticulum; G, Golgi; M, mitochondrion; N, nucleus; NE, nuclear envelope. Scale bars – (a, b): 5 µm, (c, d): 500 nm, (e–g): 1 µm. Reprinted by permission from Peddie et al., 2014 © Elsevier.
Figure 2. Overview of CLEM labels. Several CLEM labels have been developed over the years. Here, we have composed an overview of the labels discussed and have divided them into two categories: immuno‐ and genetic labels. Any label in this overview has successfully been used for CLEM experiments, but each has its advantages and disadvantages. Which label can be used varies for each project and depends on the biological question and feasibility. In general, a label that ensures optimal imaging in both LM and EM should be chosen.
Figure 3. Double‐labelled probes for visualisation in LM and EM. (a) Stills from a movie taken after 5 min of internalisation of both Tf–Alexa594–5 nm Au and EGF–Alexa488–10 nm Au (time indicated in seconds). The arrow points to a structure where the probes are initially colocalised. At time point 0, there appears to be segregation between the red fluorescence on the left and green on the right. At this point, the sample was taken and high‐pressure frozen and processed for EM. (b) To study whether the apparent segregation of the probes in the light microscope was true, the same cell was retraced in the EM. (c) An overlay of the last fluorescent frame onto this EM overview helps to identify the region of the cell where the structure of interest is located (boxed area in (b)). The structure of interest indeed shows segregation in the distribution of Tf–Alexa594–5 nm Au (red arrows) and EGF–Alexa488–10 nm Au (green arrows). Reprinted from Brown and Verkade, © US National Library of Medicine National Institutes of Health.
Figure 4. Photooxidation of Alexa dyes. (a, b) Endocytic uptake of Alexa 568‐labelled high‐density lipoproteins. HepG2 cells, on day 3 after seeding, were incubated with 50 µg mL–1 of high‐density lipoprotein Alexa 568 for 1 h. Cells, washed twice with PBS, were fixed and illuminated for 20 min utilising a TRITC filter. The intensely stained organelles in the light microscope (a) after photooxidation are identified as multivesiculated bodies at the EM level (b). (c, d) Chinese hamster ovary cells transiently transfected with N‐acetylgalactosaminyltransferase‐1 (GalNacT‐1). The cDNA sequence from GalNacT‐1 was cloned into the mammalian expression vector pEGFP‐N (Clontech Laboratories, Mountain View, CA, USA) with the GFP on the C‐terminus. Twenty‐four hours after transfection, the cells were fixed and illuminated for 30 min using a FITC filter setting. (c) Fluorescence light microscopy shows the Golgi resident GalNAcT‐1GFP localised to a juxta‐nuclear region. (d) After photooxidation, the DAB deposits are localised to cisternal lumina at one side of the Golgi stack. Reprinted by permission from Meisslitzer‐Ruppitsch et al., © John Wiley and Sons.
Figure 5. APEX, the GFP for EM? (a) EM images of HEK cells stably expressing MICU1‐APEX2. Two fields of view are shown. (b) Mitochondria of untransfected HEK cells processed under identical conditions. Scale bars–500 nm. Reprinted by permission from Lam et al., © Nature Publishing Group.
Figure 6. Using NIRB to approach and delineate ROI in 3D‐SEM. (a) Schematic illustrating the location of the ROI. (b) Maximum intensity projection of a confocal image stack in the ROI. (c) Fiducial branding marks applied to the ROI by NIRB and visualised by their autofluorescence. Arrows indicate horizontal and vertical marks delimiting the ROI, as burned into the tissue a few micrometers more ventral than the position of the neuron of interest. Two additional horizontal marks were applied adjacent to the DC ms axon branch point, ‘clasping’ it at the same ventral–dorsal depth (outlined in green). (d) The strategy for approaching and quickly finding the ROI in the resin‐embedded VNC. A guiding line is branded from the anterior end of the tissue to close to the ROI (long yellow line in magnified inset). This mark can be followed when cutting and imaging the neuropil from anterior to posterior in transverse direction (illustrated by grey plane and arrows). Additional smaller marks are placed to provide information about progress in the anterior–posterior directions (smaller vertical lines). (e) Branding marks (modelled in (d)) visualised by autofluorescence. (f) EM image of a transverse section during approach of the ROI, at the position corresponding to dotted lines in magnified (d) and (e). Branding marks are promptly identified in the tissue (arrowheads, numbers as in (d)). (g) Single confocal section in the plane of the neuronal branch point, clasped by two horizontal branding marks (arrows). (h) EM image of a transverse section in which the ms axon (pseudocoloured red) is clasped by the two horizontal branding marks (yellow). (h′) A magnification of the boxed area in (h), showing that the ultrastructure of cellular components, such as axonal microtubules (arrowheads) and synaptic vesicles (asterisk), is well preserved even in close proximity to the branding mark. (i) 3D reconstruction of the ms axon (red) and the branding marks (yellow) segmented from the stack of EM images in the ROI. Identity of the neuron is confirmed by correlating its morphology and relative position to the marks with the light microscopy data (c). Body axes are indicated in (b), (e–i); a, anterior; d, dorsal; p, proximal. Scale bars – (b): 10 µm, (e): 50 µm, (f): 2 µm, (g, h): 5 µm, (h′): 1 µm. Reprinted by permission from Urwyler et al. (). Open Access article distributed under the terms of the Creative Commons License Attribution 3.0 Unported.


Adams SR, Campbell RE, Gross LA, et al. (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. Journal of the American Chemical Society 124: 6063–6076.

Agronskaia AV, Valentijn JA, van Driel LF, et al. (2008) Integrated fluorescence and transmission electron microscopy. Journal of Structural Biology 164: 183–189.

Bell K, Mitchell S, Paultre D, Posch M and Oparka K (2013) Correlative imaging of fluorescent proteins in resin‐embedded plant material. Plant Physiology 161: 1595–1603.

Bishop D, Nikic I, Brinkoetter M, et al. (2011) Near‐infrared branding efficiently correlates light and electron microscopy. Nature Methods 8: 568–570.

Boassa D (2015) Correlative microscopy for localization of proteins in situ: pre‐embedding immuno‐electron microscopy using FluoroNanogold, gold enhancement, and low‐temperature resin. Methods in Molecular Biology 1318: 173–180.

Böttcher B (2012) Transmission Electron Microscopy: Preparation of Specimens. eLS. Chichester: John Wiley & Sons Ltd. DOI: 10.1002/9780470015902.a0002998.pu.

Brown E, Mantell J, Carter D, Tilly G and Verkade P (2009) Studying intracellular transport using high‐pressure freezing and Correlative Light Electron Microscopy. Seminars in Cell & Developmental Biology 20: 910–919.

Brown E and Verkade P (2010) The use of markers for correlative light electron microscopy. Protoplasma 244: 91–97.

Bruchez M Jr Moronne M, Gin P, Weiss S and Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281: 2013–2016.

Chan WC and Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281: 2016–2018.

Deerinck TJ, Martone ME, Lev‐Ram V, et al. (1994) Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. The Journal of Cell Biology 126: 901–910.

Deerinck TJ, Giepmans BN, Smarr BL, Martone ME and Ellisman MH (2007) Light and electron microscopic localization of multiple proteins using quantum dots. Methods in Molecular Biology 374: 43–53.

Denk W and Horstmann H (2004) Serial block‐face scanning electron microscopy to reconstruct three‐dimensional tissue nanostructure. PLoS Biology 2: e329.

Eberle AL, Mikula S, Schalek R, et al. (2015) High‐resolution, high‐throughput imaging with a multibeam scanning electron microscope. Journal of Microscopy 259: 114–120.

Ellisman MH, Deerinck TJ, Shu X and Sosinsky GE (2012) Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging. Methods in Cell Biology 111: 139–155.

Fritschy JM and Härtig W (2001) Immunofluorescence. eLS. Chichester: John Wiley & Sons Ltd.

Gaietta GM, Deerinck TJ and Ellisman MH (2011) Correlated live cell light and electron microscopy using tetracysteine tags and biarsenicals. Cold Spring Harbor Protocols 2011: pdb.top94.

Giepmans BN, Deerinck TJ, Smarr BL, Jones YZ and Ellisman MH (2005) Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nature Methods 2: 743–749.

Gilerovitch HG, Bishop GA, King JS and Burry RW (1995) The use of electron microscopic immunocytochemistry with silver‐enhanced 1.4‐nm gold particles to localize GAD in the cerebellar nuclei. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society 43: 337–343.

Godman GC, Morgan C, Breitenfeld PM and Rose HM (1960) A correlative study by electron and light microscopy of the development of type 5 adenovirus. II. Light microscopy. The Journal of Experimental Medicine 112: 383–402.

Grabenbauer M, Geerts WJ, Fernadez‐Rodriguez J, et al. (2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nature Methods 2: 857–862.

Griffin BA, Adams SR and Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 10 (5374): 269–272.

Härtig W and Fritschy JM (2001) Indirect Immunofluorescence of Tissues. eLS. Chichester: John Wiley & Sons Ltd.

Härtig W and Fritschy JM (2009) Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies. eLS. Chichester: John Wiley & Sons Ltd.

Hohn K, Fuchs J, Frober A, et al. (2015) Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy. Journal of Microscopy 259: 121–128.

Ishitani T, Hirose H and Tsuboi H (1995) Focused‐ion‐beam digging of biological specimens. Journal of Electron Microscopy 44: 110–114.

Jimenez‐Banzo A, Nonell S, Hofkens J and Flors C (2008) Singlet oxygen photosensitization by EGFP and its chromophore HBDI. Biophysical Journal 94: 168–172.

Joensuu M, Belevich I, Ramo O, et al. (2014) ER sheet persistence is coupled to myosin 1c‐regulated dynamic actin filament arrays. Molecular Biology of the Cell 25: 1111–1126.

Knott GW, Holtmaat A, Trachtenberg JT, Svoboda K and Welker E (2009) A protocol for preparing GFP‐labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nature Protocols 4: 1145–1156.

Knott G, Rosset S and Cantoni M (2011) Focussed ion beam milling and scanning electron microscopy of brain tissue. Journal of Visualized Experiments 53: e2588.

Kobayashi K, Cheng D, Huynh M, et al. (2012) Imaging fluorescently labeled complexes by means of multidimensional correlative light and transmission electron microscopy: practical considerations. Methods in Cell Biology 111: 1–20.

Kroese F (2001) Immunohistochemical Detection of Tissue and Cellular Antigens. eLS. Chichester: John Wiley & Sons Ltd.

Lam SS, Martell JD, Kamer KJ, et al. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nature Methods 12: 51–54.

Liv N, Zonnevylle AC, Narvaez AC, et al. (2013) Simultaneous correlative scanning electron and high‐NA fluorescence microscopy. PLoS One 8: e55707.

Maranto AR (1982) Neuronal mapping: a photooxidation reaction makes Lucifer yellow useful for electron microscopy. Science 217: 953–955.

Martell JD, Deerinck TJ, Sancak Y, et al. (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology 30: 1143–1148.

Masters B (2009) History of the Electron Microscope in Cell Biology. eLS. Chichester: John Wiley & Sons Ltd.

Meisslitzer‐Ruppitsch C, Rohrl C, Neumuller J, Pavelka M and Ellinger A (2009) Photooxidation technology for correlated light and electron microscopy. Journal of Microscopy 235: 322–335.

Micheva KD and Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55: 25–36.

Modla S and Czymmek KJ (2011) Correlative microscopy: a powerful tool for exploring neurological cells and tissues. Micron 42: 773–792.

Muller‐Reichert T and Verkade P (2012) Introduction to correlative light and electron microscopy. Methods in Cell Biology 111: xvii–xix.

Orrit M (2014) Nobel Prize in Chemistry: celebrating optical nanoscopy. Nature Photonics 8: 887–888.

Peddie CJ, Blight K, Wilson E, et al. (2014) Correlative and integrated light and electron microscopy of in‐resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy 143: 3–14.

Polishchuk RS, Polishchuk EV, Marra P, et al. (2000) Correlative light‐electron microscopy reveals the tubular‐saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. The Journal of Cell Biology 148: 45–58.

Robinson JM and Vandre DD (1997) Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: an important tool for correlative microscopy. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society 45: 631–642.

Robinson JM, Takizawa T, Vandre DD and Burry RW (1998) Ultrasmall immunogold particles: important probes for immunocytochemistry. Microscopy Research and Technique 42: 13–23.

Sandell JH and Masland RH (1989) Shape and distribution of an unusual retinal neuron. The Journal of Comparative Neurology 280: 489–497.

Schwarz H and Hohenberg H (2001) Immuno‐electron Microscopy. eLS. Chichester: John Wiley & Sons Ltd.

Seliger RL and Fleming WP (1974) Focused ion beams in microfabrication. Journal of Applied Physics 45: 1416–1422.

Shu X, Lev‐Ram V, Deerinck TJ, et al. (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biology 9: e1001041.

Studer D, Humbel BM and Chiquet M (2008) Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochemistry and Cell Biology 130: 877–889.

Urwyler O, Izadifar A, Dascenco D, et al. (2015) Investigating CNS synaptogenesis at single‐synapse resolution by combining reverse genetics with correlative light and electron microscopy. Development 142: 394–405.

Watanabe S, Punge A, Hollopeter G, et al. (2011) Protein localization in electron micrographs using fluorescence nanoscopy. Nature Methods 8: 80–84.

Further Reading

Blazquez‐Llorca L, Hummel E, Zimmerman H, et al. (2015) Correlation of two‐photon in vivo imaging and FIB/SEM microscopy. Journal of Microscopy 259: 129–136.

Gibson KH, Vorkel D, Meissner J and Verbavatz JM (2014) Fluorescing the electron: strategies in correlative experimental design. Methods in Cell Biology 124: 23–54.

Goetz JG, Monduc F, Schwab Y and Vermot J (2015) Using correlative light and electron microscopy to study zebrafish vascular morphogenesis. Methods in Molecular Biology 1189: 31–46.

Kim D, Deerinck TJ, Sigal YM, et al. (2015) Correlative stochastic optical reconstruction microscopy and electron microscopy. PLoS One 10: e0124581.

Kremer A, Lippens S, Bartunkova S, et al. (2015) Developing 3D SEM in a broad biological context. Journal of Microscopy 259: 80–96.

Loussert C, Forestier CL and Humbel BM (2012) Correlative light and electron microscopy in parasite research. Methods in Cell Biology 111: 59–73.

Maco B, Holtmaat A, Cantoni M, et al. (2013) Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons. PLoS One 8: e57405.

Peddie CJ and Collinson LM (2014) Exploring the third dimension: volume electron microscopy comes of age. Micron 61: 9–19.

Peddie CJ, Liv N, Hoogenboom JP and Collinson LM (2014) Integrated light and scanning electron microscopy of GFP‐expressing cells. Methods in Cell Biology 124: 363–389.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Kremer, Anna, Lippens, Saskia, and Guérin, Christopher J(Jan 2016) Correlative Light and Electron Microscopy: Methods and Applications. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025983]